Angiotensin-(1-7) inhibits vascular calcification in rats

Peptides 42 (2013) 25–34

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Angiotensin-(1-7) inhibits vascular calcification in rats Yu-Bin Sui a , Jin-Rui Chang b , Wen-Jia Chen a , Lei Zhao b , Bao-Hong Zhang d , Yan-Rong Yu b,c , Chao-Shu Tang b,c , Xin-Hua Yin a,∗ , Yong-Fen Qi b,c,∗∗ a

Department of Cardiology, the First Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang, China Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100191, China Laboratory of Cardiovascular Bioactive Molecule, School of Basic Medical Sciences, Peking University, Beijing 100191, China d Capital Institute of Pediatrics, Beijing 100020, China b c

a r t i c l e

i n f o

Article history: Received 14 September 2012 Received in revised form 13 December 2012 Accepted 13 December 2012 Available online 3 January 2013 Keywords: Angiotensin-(1-7) Vascular calcification Angiotensin converting enzyme 2 (ACE2) Mas receptor

a b s t r a c t Angiotensin-(1-7) [Ang-(1-7)] is a new bioactive heptapeptide in the renin–angiotensin–aldosterone system (RAAS) with potent protective effects in cardiovascular diseases, opposing many actions of angiotensin II (Ang II) mediated by Ang II type 1 (AT1) receptor. It is produced mainly by the activity of angiotensin-converting enzyme 2 (ACE2) and acts through the Mas receptor. However, the role of Ang-(1-7) in vascular calcification (VC) is still unclear. In this study, we investigated the protective effects of Ang-(1-7) on VC in an in vivo rat VC model induced by vitamin D3 plus nicotine. The levels of ACE2 and the Mas receptor, as well as ACE, AT1 receptor, Ang II type 2 receptor and angiotensinogen, were significantly increased in calcified aortas, and Ang-(1-7) reversed the increased levels. Ang-(1-7) restored the reduced expression of lineage markers, including smooth muscle (SM) ␣-actin, SM22␣, calponin and smoothelin, in vascular smooth muscle cells (VSMCs) and retarded the osteogenic transition of VSMCs by decreasing the expression of bone-associated proteins. It reduced alkaline phosphatase activity and calcium deposition in VC and alleviated the hemodynamic disorders of rats with VC. We provide the first in vivo evidence that Ang-(1-7) can inhibit the development of VC by inhibiting the osteogenic transition of VSMCs, at least in part by decreasing levels of the ACE/Ang II/AT1 axis. The increased expression of ACE2 and the Mas receptor in calcified aortas suggests the involvement of the ACE2/Ang-(1-7)/Mas axis during VC. Ang-(1-7) might be an efficient endogenous vasoprotective factor for VC. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Vascular calcification (VC) is a common complication for patients with advanced atherosclerosis, diabetes mellitus or chronic kidney disease (CKD), especially those with end-stage renal disease (ESRD) on hemodialysis [1,18,25]. The metabolic disorders of altered phosphate, calcium, parathyroid hormone and vitamin D levels are involved in the pathogenesis of VC [19,27,33]. VC is an important contributor to cardiovascular morbidity and mortality and considered a strong prognostic marker [20,23,31]. Recent studies have revealed that VC is an active, cell-mediated and highly regulated process resembling bone mineralization [38]. The mechanisms of VC involve gain of osteogenic induction and

∗ Corresponding author. Tel.: +86 451 85555063; fax: +86 451 86620586. ∗∗ Corresponding author at: Laboratory of Cardiovascular Bioactive Molecule, School of Basic Medical Sciences, Peking University, Beijing 100191, China. Tel.: +86 10 82805627; fax: +86 10 82805627. E-mail addresses: [email protected] (X.-H. Yin), [email protected] (Y.-F. Qi). 0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.12.023

loss of osteogenic inhibition. The disruption of the balance in favor of osteogenic promoters will induce changes in the phenotype of vascular smooth muscle cells (VSMCs) transforming them into osteoblast-like phenotype [8]. The phenotypic transformation of VSMCs is accompanied by decreased expression of contractile markers, including SM ␣-actin, SM22␣, calponin and smoothelin, as well as increased expression of bone-associated factors such as alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP2), osteopontin (OPN) and osteocalcin (OCN), and transcription factors such as core binding factor ␣ 1 (Cbf␣1) and osterix [32,37,40]. Although the current treatment for VC is preventive and based on reducing hyperphosphatemia and hypercalcemia, new specific treatments for VC should be investigated. All the processes involved in VC, especially systemic and local factors that can promote or inhibit VC, are potential therapeutic targets [29]. In recent years, numerous studies, including our previous work, have shown that endogenous cardiovasoactive peptides are involved in arterial calcification. Some vasodilator peptides, such as intermedin, adrenomedullin and cortistatin, inhibit VC [5,6,22,30], whereas some vasoconstrictor peptides, such as endothelin-1 and angiotensin II (Ang II), promote VC [14,17,45]. Hence, further

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investigation of endogenous cardiovasoactive substances may reveal new therapeutic strategies for VC. The heptapeptide angiotensin-(1-7) [Ang-(1-7)] is a new bioactive component in the renin–angiotensin–aldosterone system (RAAS) [34]. Angiotensinogen (AGT) is the precursor molecule of RAAS and its cleavage product is angiotensin I (Ang I). Ang I is a substrate of angiotensin converting enzyme (ACE) and yields Ang II, which is the major component of RAAS. Ang II binds 2 receptor subtypes, type 1 (AT1) and type 2 (AT2). Most Ang II actions are mediated by AT1, whereas AT2 mediates the beneficial effects counter-regulating those of AT1 [7]. Ang-(1-7) can be formed mainly from Ang II through the activity of ACE2 [34]. ACE2, a homolog of ACE, is a carboxypeptidase that catalyzes the conversion of Ang II to Ang-(1-7) [41]. Ang-(1-7) acts through binding to the specific Mas receptor, a seven-transmemberane G proteincoupled receptor encoded by Mas1 oncogene [36]. Ang-(1-7) has been found to oppose many actions of Ang II in the heart, vessels, lungs, kidneys, brain and liver [10,35]. Thus, the RAAS is composed of 2 opposite arms: the ACE/Ang II/AT1 axis and the ACE2/Ang-(1-7)/Mas axis. The ACE2/Ang-(1-7)/Mas axis becomes the major counter-regulatory system against the ACE/Ang II/AT1 axis at both systemic and local levels [11,12,16]. As a strong vasoprotective factor, Ang-(1-7) exerts vasodilatation, anti-angiogenesis, anti-thrombosis, and anti-proliferation effects in vivo and in vitro [35]. However, the effects of Ang-(1-7) on VC still remain unclear and need to be explored. The aim of this study was to study the role of endogenous factors in VC. We examined whether Ang-(1-7) inhibits VC in a rat model of VC induced by vitamin D3 plus nicotine (VDN) and investigated changes in major members of the ACE2/Ang-(1-7)/Mas and the ACE/Ang II/AT1 axes with calcification. 2. Materials and methods 2.1. Animals and reagents Male Sprague-Dawley (SD) rats (180–200 g) were obtained from the Animal Center, Health Science Center, Peking University (Beijing). All animal care and experimental protocols complied with the Animal Management Rule of the Ministry of Health, People’s Republic of China (Document No. 55, 2001) and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Alzet Mini-Osmotic Pumps, model 2004, were from Durect Corp. (Cupertino, CA, USA). Synthetic Ang-(1-7) was from Phoenix Pharmaceuticals (Belmont, CA, USA). Vitamin D3 and nicotine were from Sigma (St. Louis, Mo, USA). The ALP assay kit was from Nanjing Jiancheng Bioengineering Company (Nanjing, Jiangsu, China), the calcium assay kit was from Biosino Bio-Technology and Science (Beijing), the ACE activity assay kit was from Ningbo Ruiyuan BioTechnology Co., Ltd (Ningbo, Zhejiang, China) and the ACE calibrator was from Audit Diagnostics (Cork, Ireland). Other chemicals and reagents were of analytical grade.

Table 1 Primers for quantitative real-time PCR. Target

Sequence

AGT

Sense Antisense

5 -TCC ACC CCT TTC ATC TCC TCT-3 5 -CTC GCA GGG TCT TCT CAT CC-3

ACE

Sense Antisense

5 -ACG TCC CGG AAA TAC GAA G-3 5 -GCA TCA GAG TAG CCG TTG AG-3

ACE2

Sense Antisense

5 -ATC TAC CCA ACA CTT AAG CCA CC-3 5 -TAC TTT CTC CTT TGC CAA TGT CC-3

AT1

Sense Antisense

5 -CTC AAG CCT GTC TAC GAA AAT GAG-3 5 -TAG ATC CTG AGG CAG GGT GAA T-3

AT2

Sense Antisense

5 -ATCTGGCTGTGGCTGACTTAC-3 5 -TTGCCAGGGATTCCTTCTC-3

Mas

Sense Antisense

5 -TTGGTGGTGAAGATACGGAAGA-3 5 -GCATGGGCATGGCAAAGAT-3

BMP2

Sense Antisense

5 -TCA AGC CAA ACACAA ACA GC-3 5 -TGA GCT AAG CTC AGT GGG-3

Cbf␣1

Sense Antisense

5 -GCC AGG TTC AAC GAT CTG AG-3 5 -GAG GCG GTC AGA AAC AAA C-3

OPN

Sense Antisense

5 -AGA CCA GCC ATG AGT CAA GTC A-3 5 -TGA AAC TCG TGG CTC TGA TGT T-3

OCN

Sense Antisense

5 -GGT GCA AAG CCC AGC GAC TCT-3 5 -GGA AGC CAA TGT GGT CCG CTA-3

␤-Actin

Sense Antisense

5 -GAG ACC TTC AAC ACC CCA GCC-3 5 -TCG GGG CAT CGG AAC CGC TCA-3

AGT, angiotensinogen; ACE, angiotensin converting enzyme; ACE2, angiotensin converting enzyme 2; AT1, angiotensin II type 1 receptor; AT2, angiotensin II type 2 receptor; Mas, the Mas receptor; BMP2, bone morphogenetic protein 2; Cbf␣1, core binding factor ␣ 1; OPN, osteopontin; OCN, osteocalcin.

Mini-Osmotic Pump, for 28 continued days. The dose and mode of delivery of Ang-(1-7) is performed as previously published studies [21,24] with minor modification. The remaining rats were the calcification control. An additional 8 rats were given saline as a vehicle control. 2.3. Measurement of hemodynamic features of rats Hemodynamic features were measured by use of the Powerlab BL-420F Biological System (Tai-Meng Biotechnological Co., Chengdu, Sichuan, China). After rats were anesthetized intraperitoneally with pentobarbital sodium (45 mg/kg), a short PE-50 catheter filled with heparin saline (500 U/mL) was inserted to a depth of about 1.5 cm towards the heart via the right carotid artery. Hemodynamic features including heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), left ventricular systolic pressure (LVSP), LV end-diastolic pressure (LVEDP) and LV peak rate of contraction (LV + dp/dtmax ) and relaxation (LV − dp/dtmax ) were monitored. After rats were killed, the heart weight/body weight ratio (HW/BW) was measured, and aortas stripped of intima and adventitia were harvested. Samples were stored at −80 ◦ C until use.

2.2. Rat model of VC and animal groups 2.4. ALP activity assay The rat model of VC was performed as described previously [6,22,26] with minor modification. In brief, rats were randomly assigned to control group (Control), VC group (Cal), and Ang-(1-7) treatment group (Cal + Ang-(1-7)). 17 rats were given vitamin D3 (300,000 IU/kg body weight, intramuscularly) plus nicotine (25 mg/kg body weight, in peanut oil, intragastrically) at 9 a.m. on day 1, and nicotine was re-administered at 6 p.m. on the same day. At 24 h later, 8 of these rats received Ang-(1-7) (24 ␮g/kg/hr) administered subcutaneously in saline through an Alzet

ALP activity in plasma and aorta was measured as described [6,22]. Abdominal aortic blood was collected and 2 mL of the sample was mixed with heparin (50 U/mL). Plasma was separated after centrifugation at 3000 rpm for 15 min at 4 ◦ C. Aortic tissues were homogenized in ice-cold buffer (20 mmol/L HEPES, 0.2% NP-40, and 20 mmol/L MgCl2 , pH 7.4). After centrifugation at 8000 rpm for 10 min, supernatant was collected. The ALP activity of plasma and tissue supernatants was measured with use of the ALP assay kit. The

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Fig. 1. Levels of angiotensinogen (AGT) and major enzymes/receptors of the renin–angiotensin–aldosterone system (RAAS) in vascular calcification (VC) in rat aortas. Quantitative real-time PCR of aortic mRNA levels of angiotensin-converting enzyme 2 (ACE2) (A) and the Mas receptor (B) in the ACE2/angiotensin-(1-7)/Mas (ACE2/Ang(1-7)/Mas) axis and ACE (C) and Ang II type 1 (AT1) receptor (D) in the ACE/angiotensin II/AT1 (ACE/Ang II/AT1) axis in VC rats. The expression of Ang II type 2 (AT2) receptor (E) and AGT (F) were also examined. Results are relative to ␤-actin. Con, control; Cal, calcification. Data are mean ± SD. n = 3. *P < 0.05 or **P < 0.01 vs. Con.

results of ALP activity in aorta were normalized to total protein, as determined by the Bradford method [3]. 2.5. Quantification of calcium content in aortas The aorta segments were first dried at 55 ◦ C and weighed, then dissolved in HNO3 , dried at 180 ◦ C and re-dissolved with a blank solution (27 nmol/L KCl and 27 ␮mol/L LaCl3 ). Calcium levels were determined by colorimetry through a reaction with o-cresolphthalein complexon and normalized to aortic dry weight. 2.6. von Kossa staining As we described previously [6,22], a 1-cm segment of the thoracic aorta was excised and fixed in 10% formalin. Tissue samples were dehydrated and embedded in paraffin, then cut into 6-␮m-thick sections. Some of the slides were stained with

hematoxylin-eosin (H&E). Other slides were dehydrated before being incubated in 1.5% silver nitrate solution for 1 h in the sunlight, then immersed in 5% sodium thiosulfate for 2 min, and finally counterstained with safranine (red staining). Slides were examined under a light microscope by 2 investigators blinded to treatment conditions. 2.7. Real-time PCR analysis Total RNA was extracted from rat aortas by use of Trizol reagent and then reverse transcribed into single-strand cDNA with MMLV and oligo (dT) 15 primer (Promega, Madison, WI, USA). The cDNA then underwent real-time PCR in a 20-␮L reaction mixture with the Mx3000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, USA). The amount of PCR products formed in each cycle was evaluated by Eva Green fluorescence. The forward and reverse PCR primers are in Table 1.

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Fig. 2. Levels of contractile phenotype markers of vascular smooth muscle cells (VSMCs) in rat aortas. Immunoblotting analysis of protein levels of smooth muscle (SM) ␣-actin (A), SM22␣ (B), calponin (C) and smoothelin (D). ␤-actin was a control for protein loading. Data are mean ± SD. n = 3. *P < 0.05 or **P < 0.01 vs. Con, # P < 0.05 or ## P < 0.01 vs. Cal.

2.8. Western blot analysis Rat aortic tissues were homogenized in a lysis buffer containing 0.1 mol/L NaCl, 0.01 mol/L Tris–HCl (pH 7.5), 1 mmol/L ethylene diamine tetraacetic acid (EDTA), and 500 KIU/mL aprotinin, then centrifuged at 3000 rpm for 15 min at 4 ◦ C. The supernatants were used as protein samples. After protein concentrations were assessed by the Bradford method, protein extracts were resuspended in a sample buffer containing 2% SDS, 2% ␤mercaptoethanol, 50 mmol/L Tris–HCl (pH 6.8), 10% glycerol and 0.05% bormophenol blue. Protein samples were resolved on 10% SDS-PAGE in running buffer containing 25 mmol/L Tris, 192 mmol/L glycine, and 0.1% SDS, then transferred to nitrocellulose membrane in transfer buffer containing 20 mmol/L Tris–HCl (pH 8.0), 150 mmol/L glycine, and 20% methanol. Non-specific proteins were blocked by incubating the membrane with 5% non-fat dry milk in TBS-T (20 mmol/L Tris–HCl (pH 6), 150 mmol/L NaCl and 0.02% Tween 20) for 1 h at room temperature with agitation. The membrane was then incubated with the primary antibodies (diluted in TBS-T): anti-␤-actin (1:4000), anti-SM ␣-actin (1:500), anti-OPN

(1:500) and anti-SM22␣ (1:1000, all Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-calponin (1:10000), anti-OCN (1:500) and anti-smoothelin (1:800, all Abcam, MA, USA) overnight at 4 ◦ C, then fluorescein-linked secondary antibody (anti-goat, anti-rabbit or anti-mouse) for 1 h at room temperature. The reaction was visualized by enhanced chemiluminescence. The films were scanned and relative density was analyzed by use of NIH Image software.

2.9. Measurement of plasma Ang II level We collected 2 mL of abdominal aortic blood and mixed it with EDTA (0.3 mol/L), dimercaprol (0.32 mol/L) and 8hydroxyquinoline (0.34 mol/L). Samples were then centrifuged at 3000 rpm for 15 min at 4 ◦ C. Plasma was separated and stored at −80 ◦ C. Radioimmunoassay was used to measure plasma Ang II with use of the Iodine [125 I] Ang II Radioimmunoassay kit (Beijing North Institute of Biological Technology). The coefficient of variation (CV) for within-run assays was <10% and for between-run assays <15%.

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Fig. 3. Levels of osteogenic transition markers of VSMCs in rat aortas. Quantitative real-time PCR of mRNA levels of bone morphogenetic protein 2 (BMP2) (A), core binding factor ␣ 1 (Cbf␣1) (B), osteopontin (OPN) (C), and osteocalcin (OCN) (D). Results are relative to ␤-actin. Immunoblotting analysis of protein levels of OPN (E) and OCN (F). ␤-actin was a control for protein loading. Data are mean ± SD. n = 3. *P < 0.05 or **P < 0.01 vs. Con, # P < 0.05 or ## P < 0.01 vs. Cal.

2.10. Measurement of plasma ACE activity A total of 2 mL blood was mixed with heparin (50 U/mL). After centrifugation at 3000 rpm for 15 min at 4 ◦ C, plasma was separated and stored at −80 ◦ C. Plasma ACE activity was measured by the kinetic kit as described [15]. The kinetics of ACE-mediated cleavage of the synthetic substrate N-[3-(2-furyl)acryloyl]-l-phenylalanylglycyl-glycine to furylacryloylphenylalanine and glycine was detected by reduced absorbance at 340 nm. The absorbance kinetics were measured by use of the DXC800 Automatic Biochemical Analyzer (Beckman Coulter, CA, USA) and standardized to a known ACE calibrator activity. 2.11. Statistical analysis GraphPad Prism 5.0 software was used for data analysis. Data are expressed as mean ± SD. Comparisons between 2 groups involved

the unpaired Student’s t test and more than 2 groups, one-way ANOVA, followed by Newman–Keuls multiple comparison test. A P < 0.05 was considered statistically significant. 3. Results 3.1. The levels of principle members of the ACE/Ang II/AT1 and the ACE2/Ang-(1-7)/Mas axes in VC in rats First, we investigated the expression of ACE2 and the Mas receptor of the ACE2/Ang-(1-7)/Mas axis in VDN-induced VC in rats. The mRNA levels of ACE2 and Mas were higher in VC aortas than control aortas, by 2.8-fold (P < 0.05) and 4.5-fold (P < 0.05), respectively (Fig. 1A and B). We tested the levels of ACE and AT1 receptor of the ACE/Ang II/AT1 axis, which might accelerate VC. The mRNA levels of ACE and AT1 were about 3-fold (P < 0.01) and 3.6-fold (P < 0.05) higher, respectively, in calcified than control aortas (Fig. 1C and D).

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Fig. 4. Levels of the ACE/Ang II/AT1 axis with Ang-(1-7) treatment in rat aortas and plasma. Quantitative real-time PCR of mRNA levels of ACE (A), AT1 receptor (B), AT2 receptor (C) and AGT (D) in aortas. Results are relative to ␤-actin levels. Radioimmunoassay of plasma level of Ang II (E), and enzymatic kinetic assay of plasma ACE activity (F). Data are mean ± SD. n = 3 in A–D; n = 6 in E and F. *P < 0.05 or **P < 0.01 vs. Con, # P < 0.05 or ## P < 0.01 vs. Cal.

The level of AT2 receptor, a cardiovasoprotective receptor for Ang II, was also higher by 5-fold in VC aortas (P < 0.05) (Fig. 1E). As well, we detected the level of AGT, the precursor molecule of RAAS, and found the level upregulated by about 4.5-fold in VC aortas (P < 0.01) (Fig. 1F). Thus, the levels of aortic ACE2 and Mas receptor were significantly increased during VC in rat aortas, which indicated the involvement of the ACE2/Ang-(1-7)/Mas axis in calcification injury. 3.2. Ang-(1-7) retarded the contractile VSMC phenotype transforming into an osteoblast-like phenotype in calcified rat aortas The phenotypic transformation of VSMCs into osteoblastic cells in VC involves the induction and activation of osteogenic proteins and the decrease in SMC lineage markers. As compared with control

aortas, VDN aortas showed significantly decreased protein levels of contractile phenotype marker genes, including SM ␣-actin, SM22␣, calponin and smoothelin, by 26.3% (P < 0.05), 65.9% (P < 0.01), 54.2% (P < 0.05) and 71.9% (P < 0.01), respectively. The levels were rescued by Ang-(1-7) treatment, with increases of 1.4-fold (P < 0.05), 3.6fold (P < 0.01), 2-fold (P < 0.05) and 2.6-fold (P < 0.05), respectively, as compared with VC alone (Fig. 2A–D). We further investigated the expression of osteogenic marker genes: the mRNA levels of BMP2, Cbf␣1, OPN and OCN were about 5.1-fold, 6.1-fold, 2.8-fold and 2.6-fold (all P < 0.05) higher, respectively, in calcified than control aortas; however, the upregulated expression of these genes was downregulated with Ang-(1-7) treatment, by 80.5% (P < 0.01), 82.9% (P < 0.05), 60.4% (P < 0.05) and 82.6% (P < 0.01), respectively (Fig. 3A–D). On western blot analysis, the protein levels of OPN and OCN were increased in calcified VSMCs

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by 51% and 49% (both P<0.05), and Ang-(1-7) had an inhibitory effect on OPN and OCN expression, with a 39.3% (P < 0.05) and 48.7% (P < 0.01) decrease, respectively, in protein level (Fig. 3E and F). 3.3. Ang-(1-7) reversed the elevated levels of the ACE/Ang II/AT1 axis in VC Because of the counter-balance between the 2 opposing arms of RAAS, we detected the expression of the main enzyme and receptor in the ACE/Ang II/AT1 axis with Ang-(1-7) treatment. Real-time PCR revealed decreased mRNA expression of ACE and AT1, as well as reduced AGT level, in Ang-(1-7)-treated aortas, by 61.5% (P < 0.01), 72.5% (P < 0.05) and 79.2% (P < 0.01), respectively, as compared with VC alone (Fig. 4A, B and D). Although AT2 is a vasoprotective receptor, its mRNA level was still downregulated, by 82.7% (P < 0.05), with Ang-(1-7) treatment (Fig. 4C). As well, we tested Ang II level and ACE activity in rat plasma. Ang II level and ACE activity was higher but not significantly in plasma of VC than control rats. With Ang-(1-7), Ang II level was decreased but not significantly in plasma. Interestingly, the ACE activity was lesser in Ang-(1-7) treatment group than that in VC treatment alone, by 18.6% (P < 0.01) (Fig. 4E and F). Therefore, Ang-(1-7) inhibited plasma ACE activity, which might result in the reduction of circulating Ang II concentration. 3.4. Ang-(1-7) treatment attenuated VC Compared with control rats, VC rats showed increased ALP activity, both in plasma and aortas, by 38.7% and 93.5% (both P < 0.05), respectively. With Ang-(1-7) treatment, ALP activity in plasma and aortas was reduced by 34.4% and 36.9% (both P < 0.05), respectively, as compared with VC alone (Fig. 5A and B). The calcium content was approximately 2.2-fold (P < 0.01) higher in VC than control aortas. As compared with VC alone, Ang-(1-7) treatment decreased the calcium content significantly, by 49.8% (P < 0.01) (Fig. 5C). Decreased calcium-phosphate salt deposition in Ang-(17)-treated VC aortas was further confirmed by von Kossa staining. Dispersed calcified nodules (black–brown areas) were among the elastic lamina of the tunica media in calcified arteries and were significantly reduced with Ang-(1-7) treatment (Fig. 6A). In addition, H&E staining revealed thickened vessel walls and disordered elastic fibers in calcified aortas as compared with control aortas; however, Ang-(1-7) treatment restored the vascular structure (Fig. 6B). VC is associated with fragmented and reduced elastic fibers in the vessel wall, causing lower vascular elasticity, arterial hypertension, and vascular stiffening [43]. To investigate the hemodynamic contribution of Ang-(1-7) to VC, we measured the hemodynamic parameters of rats. Compared with control rats, VC rats showed significantly increased SBP, DBP, MBP and LVSP, by 15.4% (P < 0.01), 39.8% (P < 0.01), 29.6% (P < 0.01) and 8.5% (P < 0.05), respectively (Table 2). However, Ang-(1-7) treatment attenuated VDN-induced hemodynamic disorders: SBP, DBP, MBP and LVSP were lower, by 11.7% (P < 0.05), 21.2% (P < 0.01), 19.5% (P < 0.01) and 10% (P < 0.01), respectively, as compared with VC alone (Table 2). 4. Discussion In this study, we found increased expression of ACE2 and the Mas receptor in rat aortas during VC, along with upregulated expression of AGT, ACE and AT1. Besides the pro-calcification element of the ACE/Ang II/AT1 axis, the ACE2/Ang-(1-7)/Mas axis might also be involved in this pathological process. Using a gainof-function strategy, we provide the first in vivo evidence that systemic administration of Ang-(1-7) had a marked protective effect on arterial calcification by restoring the contractile phenotype of VSMCs and inhibiting vascular osteogenesis. Our data

Fig. 5. Alkaline phosphatase (ALP) activity in rat plasma or aortas and calcium content in aortas. Effect of Ang-(1-7) on ALP activity in plasma (A) and in aortas (B), and calcium content in aortas (C). Data are mean ± SD. n = 6 in each group in A and B; n = 7 in Con, Cal + Ang-(1-7) and n = 6 in Cal in C. *P < 0.05 or **P < 0.01 vs. Con, # P < 0.05 or ## P < 0.01 vs. Cal.

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Fig. 6. Calcium deposition and vascular remodeling in rat aortas. von Kossa staining (A) of vascular calcium deposition (black/brown areas) and hematoxylin-eosin (H&E) staining (B) of vascular structure. Magnification 200× in A(a)–(c) and in B(a)–(c); magnification 400× in A(d)–(f) and in B(d)–(f).

suggest that the exogenous Ang-(1-7) could inhibit VC at least in part by decreasing the levels of the endogenous ACE/Ang II/AT1 axis, the inhibition of which has been shown to attenuate the progression of VC [2,28]. Our rat model treated with high dose of vitamin D3 plus nicotine showed typical VC, sharing similarities with calcification in human athero- and arteriosclerosis [26]. The VDN-rats showed increased calcium deposition and ALP activity in aortas, hemodynamic

disorders, and pathological remodeling of vessel walls, which agreed with previous studies [6,22]. However, Ang-(1-7) treatment significantly ameliorated these complications, which indicates the potent preventive effects of Ang-(1-7) on arterial calcification. VC is a well-documented active and regulated process, during which VSMCs undergo the transition into osteoblast-like cells [38]. In this study, Ang-(1-7) treatment prevented the loss of lineage markers of VSMCs and attenuated the osteogenic transition of

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Table 2 Hemodynamic features of rats. Control (n = 8) HW/BW (mg/g) HR (beats/min) SBP (mmHg) DBP (mmHg) MBP (mmHg) LVSP (mmHg) LVEDP (mmHg) LV + dp/dtmax (mmHg/s) LV − dp/dtmax (mmHg/s)

2.72 424.2 113.3 70.8 84.9 117.4 1.343 6667.5 −4287.7

± ± ± ± ± ± ± ± ±

0.59 79.5 9.9 15.0 12.9 6.8 3.658 807.6 836.0

Calcification (n = 9) 2.762 435.3 130.7 99.0 110.0 127.4 1.594 6737.8 −5115.8

± ± ± ± ± ± ± ± ±

0.17 44.0 10.3** 10.2** 9.8** 5.9* 2.885 767.7 612.5

Calcification + Angiotensin(1-7) (n = 8) 2.74 424.2 115.4 78.0 90.5 114.6 1.29 6434.5 −4434.9

± ± ± ± ± ± ± ± ±

0.15 84.0 9.8# 12.8## 11.7## 6.8## 2.07 829.8 857.0

Data are mean ± SD. Control (Con): rats with solvents; Calcification (Cal): rats with vascular calcification (VC) induced by vitamin D3 and nicotine; Calcification + Angiotensin(1-7) (Cal + Ang-(1-7)): VC rats with exogenous Ang-(1-7) (24 ␮g/kg/hr). HW, heart weight; BW, body weight; HR, heart rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure; LVSP, left ventricular systolic pressure; LVEDP, LV end-diastolic pressure; LV ± dp/dtmax , LV peak rate of contraction and relaxation. * P < 0.05 vs. Con. ** P < 0.01 vs. Con. # P < 0.05 vs. Cal. ## P < 0.01 vs. Cal.

VSMCs by decreasing the expression of bone-associated proteins in VDN-treated aortas. Concurrently, Ang-(1-7) decreased calcium content and decreased calcium-phosphate salt deposition in aortas of rats with VC. These results suggest that Ang-(1-7) prevented the phenotypic transition of VSMCs into osteoblasts, thus attenuated the development of VC. Conventional concepts of RAAS have to be changed by the recent discovery that ACE2 degrades Ang II generating Ang-(1-7), which acts as an endogenous antagonist of Ang II [35]. The ACE2/Ang-(17)/Mas axis is found to be a novel principal counter-regulator of the traditional ACE/Ang II/AT1 axis within RAAS, and cardiovascular homeostasis is a result of balanced activities of these 2 arms [11]. In this study, we found increased levels of ACE2 and Mas in calcified aortas, as well as increased expression of AGT, ACE and AT1. Our data suggest that the ACE2/Ang-(1-7)/Mas axis might be involved in the development of VC and that the ACE/Ang II/AT1 axis was activated and might promote ossification in VC. ACE2 expression is upregulated in cardiovascular diseases [4,13,39], and Mas expression is responsive to different injury stimuli [9]. In view of the verified beneficial effects of ACE2/Ang-(1-7)/Mas against ACE/Ang II/AT1 in physiological and pathological stimuli [12,16], the upregulation of aortic ACE2 and Mas in the calcified artery might be a response to the vascular injury. The endogenous ACE2/Ang-(17)/Mas axis might play a negative regulatory role and to some degree antagonize the opposite axis in VC. Our results showed that Ang-(1-7) reversed the increased expression of ACE/Ang II/AT1 and inhibited plasma ACE activity effectively, which might result in the reduction of circulating Ang II concentration. The results of the downregulation of ACE/Ang II/AT1 and the recovery of vascular damage with Ang-(1-7) treatment were in line with previous studies, which showed the same counteraction of 2 axes with ACE2 over-expression [46]. We also found the elevated expression of AT2 in VC. The increased level of AT2 was likely to be a counteracting response to the Ang II/AT1 over-activation and calcification injury, which agreed with the upregulation of AT2 in other cardiovascular diseases [42]. The expression of pathogenic ACE/Ang II/AT1 axis was significantly decreased in calcified rat aorta treated with Ang-(1-7). These results demonstrate that exogenous Ang(1-7) could protect vessels against calcification, at least in part by marked inhibitory effects on the ACE/Ang II/AT1 axis. It has been reported that the interaction between AT1 and AT2 plays an important role in sustaining cardiovascular homeostasis [44]. The RAAS is an auto-balanced system in which the counter-regulation of the cascade of multiple functional members is complicated and precise [12,16]. The balance of RAAS is important to sustain cardiovascular homeostasis and to prevent cardiovascular diseases. Therefore, the interaction of these 2 arms needs to be further investigated. The

limitation of our study is that we did not detect levels of Ang-(17). The molecular mechanisms and signal pathways involved in the protective effects of Ang-(1-7) on VC should be further investigated. In summary, we provide the first in vivo evidence that Ang-(17) attenuated the development of VC by retarding the osteogenic transition of VSMCs, at least in part by decreasing levels of the ACE/AngII/AT1 axis. As well, we show the upregulated expression of ACE2 and Mas in calcified arteries, which suggests the involvement of the ACE2/Ang-(1-7)/Mas axis during the pathogenesis of VC. Ang-(1-7) might be an efficient endogenous vasoprotective factor for VC, and the ACE2/Ang-(1-7)/Mas axis could become a new target for the prevention and therapy of VC. Acknowledgements This work was supported by Heilongjiang Outstanding Youth Science Fund (No. JC201001 to Xin-Hua Yin), the National Nature Science Foundation of China (No. 81270407 to Yong-Fen Qi) and Leading Academic Discipline Project of Beijing Education Bureau to Yong-Fen Qi. We are grateful for technical support from Li Chen and Qiang Shen (Peking University Health Science Center) for histochemical staining. References [1] Alexopoulos N, Raggi P. Calcification in atherosclerosis. Nat Rev Cardiol 2009;6(11):681–8. [2] Armstrong ZB, Boughner DR, Drangova M, Rogers KA. Angiotensin II type 1 receptor blocker inhibits arterial calcification in a pre-clinical model. Cardiovasc Res 2011;90(1):165–70. [3] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. [4] Burrell LM, Risvanis J, Kubota E, Dean RG, MacDonald PS, Lu S, et al. Myocardial infarction increases ACE2 expression in rat and humans. Eur Heart J 2005;26(4):369–75. [5] Cai DY, Yu F, Jiang W, Jiang HF, Pan CS, Qi YF, et al. Adrenomedullin(27–52) inhibits vascular calcification in rats. Regul Pept 2005;129(1–3):125–32. [6] Cai Y, Xu MJ, Teng X, Zhou YB, Chen L, Zhu Y, et al. Intermedin inhibits vascular calcification by increasing the level of matrix ␥-carboxyglutamic acid protein. Cardiovasc Res 2010;85(4):864–73. [7] Carey RM. Update on the role of the AT2 receptor. Curr Opin Nephrol Hypertens 2005;14(1):67–71. [8] Chillon JM, Mozar A, Six I, Maizel J, Bugnicourt JM, Kamel S, et al. Pathophysiological mechanisms and consequences of cardiovascular calcifications: role of uremic toxicity. Ann Pharm Fr 2009;67(4):234–40. [9] Dias-Peixoto MF, Ferreira AJ, Almeida PW, Braga VB, Coutinho DC, Melo DS, et al. The cardiac expression of Mas receptor is responsive to different physiological and pathological stimuli. Peptides 2012;35(2):196–201. [10] Ferreira AJ, Murca TM, Fraga-Silva RA, Castro CH, Raizada MK, Santos RA. New cardiovascular and pulmonary therapeutic strategies based on the Angiotensin-converting enzyme 2/angiotensin-(1-7)/mas receptor axis. Int J Hypertens 2012;2012:147825.

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